The mechanism by which a protein folds, given only the information in the primary sequence, remains one of the most difficult and challenging problems in biochemistry. The specific aims of this proposal are to approach this problem by determining the nature and structural properties of early intermediates on the folding pathway. Several methods are to be used including stopped 0flow NMR techniques. Site directed mutagenesis coupled with incorporation of fluorine labeled amino acids and a stopped flow NMR device we have built will allow examination of specific regions of a protein during folding. We will continue to study the E. coli dihydrofolate reductase by this method using different fluorine labeled amino acids. A second protein being examined is the fatty acid binding protein. We wish to make mutants that are stable intermediates to explore structure by NMR techniques. Fluorescence correlation spectroscopy will be used to examine rapid motions in wild-type and stable intermediates in the folding process. We are also using dihydrofolate reductase to examine the mechanism of action of the bacterial chaperone GroEL and effects of metals, nucleotides and GroES. Finally, we plan folding experiments with the bacterial chaperone PapD. Dihydrofolate reductase is the target for numerous chemotherapeutic, antibacterial and antiparasitic drugs. The intestinal fatty acid binding protein is one of a family of proteins that differ in ligand specificity (fatty acids, retinoids and bile salts) and tissue specificity and are found to be useful models for spatial and temporal differentiation in the gut. PapD is a chaperone for proteins that form filamentous pili in pathogenic bacteria.